Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction
| Title | Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction |
| Publication Type | Journal Article |
| Year of Publication | 2000 |
| Authors | Igarashi, J, Michel T |
| Journal | J Biol Chem |
| Volume | 275 |
| Pagination | 32363-70 |
| Date Published | Oct 13 |
| ISBN Number | 0021-9258 (Print)0021-9258 (Linking) |
| Accession Number | 10921915 |
| Keywords | *Lysophospholipids, *Receptors, Cell Surface, *Receptors, G-Protein-Coupled, Animals, Caveolae/drug effects/enzymology/*metabolism, Caveolin 1, Caveolins/genetics/*metabolism, Centrifugation, Density Gradient, COS Cells, Enzyme Activation/drug effects, Humans, Immediate-Early Proteins/*agonists/antagonists &, inhibitors/genetics/*metabolism, Nitric Oxide Synthase/*metabolism, Nitric Oxide/metabolism, Phosphorylation/drug effects, Precipitin Tests, Protein Binding/drug effects, Protein Transport/drug effects, Receptors, Lysophospholipid, Recombinant Fusion Proteins/metabolism, Signal Transduction/*drug effects, Sphingosine/*analogs & derivatives/antagonists & inhibitors/pharmacology, Transfection |
| Abstract | Plasmalemmal caveolae are membrane microdomains that are specifically enriched in sphingolipids and contain a wide array of signaling proteins, including the endothelial isoform of nitric-oxide synthase (eNOS). EDG-1 is a G protein-coupled receptor for sphingosine 1-phosphate (S1P) that is expressed in endothelial cells and has been implicated in diverse vascular signal transduction pathways. We analyzed the subcellular distribution of EDG-1 in COS-7 cells transiently transfected with cDNA constructs encoding epitope-tagged EDG-1. Subcellular fractionation of cell lysates resolved by ultracentrifugation in discontinuous sucrose gradients revealed that approximately 55% of the EDG-1 protein was recovered in fractions enriched in caveolin-1, a resident protein of caveolae. Co-immunoprecipitation experiments showed that EDG-1 could be specifically precipitated by antibodies directed against caveolin-1 and vice versa. The targeting of EDG-1 to caveolae-enriched fractions was markedly increased (from 51 +/- 11% to 93 +/- 14%) by treatment of transfected cells with S1P (5 microm, 60 min). In co-transfection experiments expressing EDG-1 and eNOS cDNAs in COS-7 cells, we found that S1P treatment significantly and specifically increased nitric-oxide synthase activity, with an EC(50) of 30 nm S1P. Overexpression of transfected caveolin-1 cDNA together with EDG-1 and eNOS markedly diminished S1P-mediated eNOS activation; caveolin overexpression also attenuated agonist-induced phosphorylation of EDG-1 receptor by >90%. These results suggest that the interaction of the EDG-1 receptor with caveolin may serve to inhibit signaling through the S1P pathway, even as the targeting of EDG-1 to caveolae facilitates the interactions of this receptor with ligands and effectors that are also targeted to caveolae. The agonist-modulated targeting of EDG-1 to caveolae and its dynamic inhibitory interactions with caveolin identify new points for regulation of sphingolipid-dependent signaling in the vascular wall. |
| URL | http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10921915 |